This invention relates to compositions and methods for treating malaria. Specifically, this invention relates to pharmaceutical compositions including spiro and dispiro trioxolanes, and methods of their use and manufacture.
Malaria is an acute and often chronic infectious disease resulting from the presence of protozoan parasites within red blood cells. Caused by single-celled parasites of the genus Plasmodium, malaria is transmitted from person to person by the bite of female mosquitos.
Although once prevalent in North America and other temperate regions of the world, today malaria occurs mostly in tropical and subtropic countries. Each year, between 400 million and 600 million people contract the disease, and 1.5 million to 2.7 million die of the disease.
Four species of Plasmodium protozoan parasites are generally responsible for malaria, including Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, and Plasmodium ovale. Of the four, Plasmodium falciparum is the most dangerous, accounting for half of all clinical cases of malaria and 90% of deaths from the disease.
The transmission of malaria begins when a female mosquito bites a human already infected with the malaria parasite. When the infected mosquito bites another human, sporozoites in the mosquito""s saliva are transferred into the blood, which then travel to the liver. In the liver, the sporozoites divide rapidly, then enter the bloodstream where they invade red blood cells. Inside these blood cells, the merozoites multiply rapidly until they cause the red blood cells to burst, releasing into the blood stream a new generation of merozoites that then infect other red blood cells.
The symptoms associated with malaria are generally associated with the bursting of the red blood cells. The destruction of the red blood cells spills wastes, toxin, and other debris into the blood. This in turn causes an intense fever that can leave the infected individual exhausted and bedridden. More severe symptoms associated with repeat infections and/or infection by Plasmodium falciparum include anemia, severe headaches, convulsions, delirium and, in some instances, death.
The treatment of malaria has been especially difficult due to the ability of malaria parasites to develop resistance to drugs. Quinine, an antimalarial compound that is extracted from the bark of the South American cinchona tree, is one of the oldest and most effective pharmaceuticals in existence. The downside to quinine is that it is short-acting, and fails to prevent disease relapses. Further, quinine is associated with side effects ranging from dizziness to deafness.
Chloroquine is a synthetic chemical similar to quinine. It became the drug of choice for malaria when it was developed in the 1940s due to its effectiveness, ease of manufacture, and general lack of side effects. However, in the last few decades, malaria parasites in many areas of the world have become resistant to chloroquine.
Mefloquine is another synthetic analog of quinine that has been used in the treatment of malaria. Malaria parasites have also developed resistance to mefloquine, however. Mefloquine is also associated with undesirable central nervous side effects in some patients, including hallucinations and vivid nightmares.
Antifolate drugs are effective against malaria parasites by inhibiting their reproduction. Although the parasites have also developed a resistance to antifolate drugs, the drugs can still be used effectively in combination with other types of antimalarials. The use of combination therapies in treating malaria has the drawbacks of being inconvenient and expensive, however.
More recent developments in the treatment of malaria have involved the use of the peroxide functional group, as exemplified by the drug artemisinin, which contains a unique 1,2,4-trioxane heterocyclic pharmacophore. The antimalarial action of artemisinin is due to its reaction with the iron in free heme molecules in the malaria parasite with the generation of free radicals leading to cellular destruction.
Although the clinically useful semisynthetic artemisinin derivatives are rapid acting and potent antimalarial drugs, they have several disadvantages including recrudescence, neurotoxicity, (Wesche et al., 1994) and metabolic instability. (White, 1994). Although many synthetic antimalarial 1,2,4-trioxanes have since been prepared (Cumming et al., 1996; Jefford, 1997), there exists a need in the art to identify new peroxide antimalarial agents, especially those which are easily synthesized, are devoid of neurotoxicity, and which possess improved pharmacokinetic properties, e.g. improved stability, oral absorption, etc.
Accordingly, it is a primary objective of the present invention to provide compositions and methods for prophylaxis and treatment of malaria using Spiro and dispiro 1,2,4-trioxolanes.
It is a further objective of the present invention to provide a composition and method for prophylaxis and treatment of malaria using Spiro and dispiro 1,2,4-trioxolanes that is nontoxic.
It is a further objective of the present invention to provide a composition and method for prophylaxis and treatment of malaria using Spiro and dispiro 1,2,4-trioxolanes that is metabolically stable and orally active.
It is yet a further objective of the present invention to provide a composition and method for prophylaxis and cost-effective treatment of malaria using Spiro and dispiro 1,2,4-trioxolanes.
It is a further objective of the present invention to provide compositions and methods for prophylaxis and treatment of malaria using Spiro and dispiro 1,2,4-trioxolanes that can be used either as stand-alone medicaments or in combination with other agents.
The method and means of accomplishing each of the above objectives as well as others will become apparent from the detailed description of the invention which follows hereafter.
The invention describes a method and composition for treating malaria with Spiro and dispiro 1,2,4-trioxolanes, their prodrugs and analogues. The trioxolanes of this invention are sterically hindered on one side of the trioxolane heterocycle in order to provide chemical and metabolic stability to the trioxolane ring for better in vivo activity. The spiro and dispiro trioxolanes are preferably sterically hindered with an unsubstituted, mono-, di-, or poly-substituted C5-C12 spiro cycloalkyl group, which is most preferably spiroadamantane. The spiro and dispiro trioxolanes also preferably include a spirocyclohexyl that is preferably functionalized or substituted at the 4-position or a spiropiperidyl ring that is functionalized or substituted at the nitrogen atom. The invention embraces achiral, achiral diastereomers, racemic mixtures, as well as enantiomeric forms of the compounds.
The trioxolanes of this invention possess excellent potency and efficacy against Plasmodium parasites, and a low degree of neurotoxicity. In addition, several of the trioxolanes are suitable for both oral and non-oral administration. Moreover, in comparison to artemisinin semisynthetic derivatives, the compounds of this invention are structurally simple, easy and inexpensive to synthesize, and can be used effectively alone or in conjunction with other antimalarials.
The present invention relates to the development of spiro and dispiro 1,2,4-trioxolanes for use in the prophylaxis and treatment of malaria. The present invention is predicated upon the unexpected discovery that trioxolanes that are relatively sterically hindered on at least one side of the trioxolane heterocycle provide metabolic and chemical stability to the trioxolane ring, thereby providing better in vivo activity, especially with respect to oral administration.
As used herein the term xe2x80x9cprophylaxis-effective amountxe2x80x9d refers to a concentration of compound of this invention that is effective in inhibiting or preventing infection and subsequent disease by malarial parasites. Likewise, the term xe2x80x9ctreatment-effective amountxe2x80x9d refers to a concentration of compound that is effective in treating malaria in terms of preventing an increase in the concentration of malarial parasites, decreasing the concentration of malarial parasites, and/or xe2x80x9ccuringxe2x80x9d a malaria infection, i.e. survival for 30 days post-infection.
Trioxolanes are relatively stable peroxidic compounds based on literature precedent (Griesbaum et al., 1997a; 1997b). This may be due, in part, to the lack of xcex1-hydrogen atoms. The present inventors have synthesized new compounds in the trioxolane class having both superior antimalarial potency and oral efficacy. Furthermore, the compounds of this invention have low toxicity, and half-lives conducive to treatment of malaria which are believed will permit short-term treatment regimens comparing favorably to other artemisinin-like drugs. These compounds may also be used in malaria prophylaxis.
The tetrasubstituted trioxolanes of this invention have the following general structural formula: 
wherein R1, R2, R3, and R4 represent combinations of ring systems, acyclic systems, and functional groups that provide sufficient steric hindrance about the trioxolane ring in order to give the ring chemical and metabolic stability. R1, R2, R3 and R4 may be the same or different, and may be a linear or branched alkyl, aryl, or alkaryl group which is optionally substituted. In the alternative, R1 and R2 taken together and/or R3 and R4 taken together may form an alicyclic group which is optionally interrupted by one or more oxygen, sulfur or nitrogen atoms and which group is optionally substituted. In no event may any of R1, R2, R3 or R4 be hydrogen.
Preferably, R1 and R2 taken together and/or R3 and R4 taken together is a mono- or di-substituted C5-C12 spirocycloalkyl group which is optionally interrupted by one or more oxygen, sulfur, or nitrogen atoms, and which group is optionally substituted.
Most preferably, R1 and R2 taken together or R3 and R4 is spiroadamantane. It is hypothesized that the sterically demanding adamantane protects the trioxolane ring from premature chemical or metabolic decomposition in situ.
The inventors have further found that in the most preferred compounds of this invention, R1 and R2 taken together is adamantane, and R3 and R4 taken together is a spirocyclohexyl ring that is functionalized or substituted at the 4-position. The spirocyclohexyl ring may be optionally interrupted by one or more oxygen, sulfur or nitrogen atoms. The functional group may be a linear or branched alkyl, ketone, acid, alcohol, amine, amide, sulfonamide, ether, ester, oxime, urea, ether, sulfone, lactone, carbamate, semicarbazone, phenyl, heterocycle, or alicyclic group which is optionally substituted. Other substituents at the 4-position of the spirocyclohexyl ring are also possible that fall within the scope of this invention. The spirocyclohexyl ring may also be substituted at other positions besides the 4-position. For instance, the inventors have synthesized several compounds substituted at the 2-position of the spirocyclohexyl ring that provide excellent antimalarial potency.
Below are several dispiro 1,2,4-trioxolanes synthesized in accordance with the teachings of this invention. xe2x80x9cOZxe2x80x9d is an internal designation for these compounds that will be used throughout the remainder of the application for convenience: 
The prototype trioxolanes of this invention are OZ03 and OZ05. Preferred compounds identified thus far include OZ03, OZ05, OZ10, OZ11, OZ15, OZ19, OZ23, OZ24, OZ25, OZ27, OZ31, OZ32, OZ40, OZ41, OZ43, OZ44, OZ47, OZ48, OZ61, OZ63, OZ70, OZ71, OZ77, OZ78, OZ80, OZ83, OZ84, and OZ89. The most preferred compounds are OZ10, OZ11, OZ15, OZ23, OZ25, OZ27, OZ31, OZ41, OZ43, OZ47, OZ63, OZ71, OZ77, OZ78, and OZ89. In general, the highest in vitro potency against malarial parasites is obtained for trioxolanes functionalized or substituted at the 4-position of the spirocyclohexyl ring. As a general rule, non-symmetrical, achiral trioxolanes, such as OZ03, OZ05, OZ10 and OZ11, OZ12, OZ13, OZ18, are also preferred.
Notable features of these spiro and dispiro 1,2,4-trioxolanes in comparison to the artemisinin semisynthetic derivatives are their structural simplicity and ease of synthesis. For example, dispiro trioxolanes may be easily synthesized by the coozonolysis of the O-methyl oximes of cycloalkanones in the presence of the requisite cycloalkanone derivatives according to the method of Griesbaum et al. (1997a; 1997b) as illustrated below for the symmetrical dispiro cyclohexyl trioxolane: 
If yields are low in this coozonolysis reaction, yields can improve dramatically when the O-methyloxime and ketone are xe2x80x9creversed.xe2x80x9d This novel procedure provides a uniquely convenient method to synthesize spiro and dispiro trioxolanes. The trioxolanes may be purified by crystallization or by flash column chromatography. Their structures and purity may be confirmed by analytical HPLC, 1H and 13C NMR, IR, melting point and elemental analysis.
The following cycloalkanone and cycloalkanedione starting materials can be obtained from Aldrich Chemical Co. or from TCI American Organic Chemicals: cyclohexanone, cycloheptanone, cyclooctanone, cyclodecanone, cyclododecanone, 1,3-cyclohexanedione, 1,4-cyclohexanedione, 2,2-dimethylcyclohexanone, 2-adamantanone, bicyclo[3.3.1]nonan-9-one, tetrahydro-4H-pyran-4-one, 1-carboethoxy-4-piperidone, 1-benzoyl-4-piperidone, 1-indanone, 2-indanone, xcex1-tetralone, xcex2-tetralone, bicyclo[3.3.1]nonan-3,7-dione, 1,4-cyclohexanedione-mono-2,2-dimethyltrimethylene ketal, cis-bicyclo[3.3.0]octane-3,7-dione, and 4-carboethoxycyclohexanone.
The cycloalkanone starting materials may also be synthesized. For instance, the inventors have synthesized 4,4-dimethylcyclohexanone and 4,4-diphenylcyclohexanone by catalytic hydrogenation (Augustine, 1958) of the commercially available enones. Also, 2-carboethoxyethylcyclohexanone was synthesized by treatment of the pyrrolidine enamine of cyclohexanone with ethyl acrylate (Stork et al., 1963). Persons skilled in the art can readily ascertain other appropriate means of synthesizing the starting materials and compounds in accordance with this invention.
Differential scanning calorimetry (DSC) experiments for trioxolanes of this invention reveal that these compounds have good thermal stability, comparable to artemisinin.
The spiro and dispiro trioxolane compositions of the present invention may be generally used for the prophylaxis and treatment of malaria. The trioxolane compositions of the present invention are administered along with a pharmaceutically acceptable carrier. Any pharmaceutically acceptable carrier may be generally used for this purpose, provided that the carrier does not significantly interfere with the stability or bioavailability of the trioxolane compounds of this invention.
The trioxolanes of this invention can be administered in any effectively pharmaceutically acceptable form to warm blooded animals, including human and other animal subjects, e.g. in topical, lavage, oral, suppository, parenteral, or infusible dosage forms, as a topical, buccal, sublingual, or nasal spray or in any other manner effective to deliver the agents. The route of administration will preferably be designed to optimize delivery and/or localization of the agents to target cells.
In addition to the active compounds i.e. the trioxolanes, the pharmaceutical compositions of this invention may contain suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Oral dosage forms encompass tablets, capsules, and granules. Preparations which can be administered rectally include suppositories. Other dosage forms include suitable solutions for administration parenterally or orally, and compositions which can be administered buccally or sublingually.
The pharmaceutical preparations of the present invention are manufactured in a manner which is itself well known in the art. For example the pharmaceutical preparations may be made by means of conventional mixing, granulating, dragee-making, dissolving, lyophilizing processes. The processes to be used will depend ultimately on the physical properties of the active ingredient used.
Suitable excipients are, in particular, fillers such as sugars for example, lactose or sucrose mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch, paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added, such as the above-mentioned starches as well as carboxymethyl starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are flow-regulating agents and lubricants, for example, such as silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate and/or polyethylene glycol. Oral dosage forms may be provided with suitable coatings which, if desired, may be resistant to gastric juices.
For this purpose concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, dyestuffs and pigments may be added to the tablet coatings, for example, for identification or in order to characterize different combination of compound doses.
Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition stabilizers may be added. Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of the active compounds with the suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols, or higher alkanols. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base material include for example liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
Suitable formulations for parenteral administration include aqueous solutions of active compounds in water-soluble or water-dispersible form. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, including for example, sodium carboxymethyl cellulose, sorbitol and/or dextran. Such compositions may also comprise adjuvants such as preserving, wetting, emulsifying, and dispensing agents. They may also be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents into the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved or suspended in sterile water, saline, or other injectable medium prior to administration.
In addition to administration with conventional carriers, active ingredients may be administered by a variety of specialized delivery drug techniques which are known to those of skill in the art, such as portable infusion pumps.
The trioxolane compositions of the present invention are administered along with a pharmaceutically acceptable carrier in an amount sufficient to prevent malarial infection and/or treat an active infection. The trioxolane compounds of this invention have extremely low toxicity and a low degree of side effects even at high doses. The dosing range of the trioxolane compositions will vary depending on a number of factors, such as whether it is used for prophylaxis or treatment of an active infection, route of administration, dosing schedule, etc. In general, the therapeutic dose of trioxolane may range between about 0.1-1000 mg/kg/day, with between about 1-100 mg/kg/day being preferred. The foregoing doses may be administered as a single dose or may be divided into multiple doses for administration. The trioxolane compositions may be administered once to several times daily. For malaria prevention, a typical dosing schedule could be, for example, 2.0-1000 mg/kg weekly beginning 1-2 weeks prior to malaria exposure taken up until 1-2 weeks post-exposure.
The spiro and dispiro trioxolanes of this invention have been found to be effective in the treatment of schistosomiasis. Schistosomiasis ranks second behind malaria in terms of socioeconomic and public health importance in tropical and subtropical areas. The disease is endemic in 74 developing countries, infecting more than 200 million people in rural agricultural and peri-urban areas. An estimated 500-600 million people worldwide are at risk from the disease.
The major forms of human schistosomiasis are caused by five species of water-borne flatworm, or blood flukes, called schistosomes. One of these species is Schistosoma mansoni, which has been reported in 53 countries in Africa, the Eastern Mediterranean, the Caribbean, and South America. The parasites enter the body through contact with infested surface water, primarily among people engaged in agriculture and fishing. The parasites normally infect the host during the cercaria, or larval stage. Once inside the host, the cercaria develop into adults or schistosomes.
Current treatments for schistosomiasis have focused primarily on prophylaxis, i.e. prevention of host infection by cercaria. Currently, praziquantel is the most widely used drug for treatment of schistosomiasis. While artemether has demonstrated activity in the prophylaxis of schistosomiasis, it has not shown any activity against adult S. mansoni. 
It has now been unexpectedly discovered that the spiro and dispiro trioxolanes of this invention are active against both cercaria and adult S. mansoni when administered in the dosages and manner outlined above with respect to treatment of malarial parasites. Preferred compounds identified for use in the treatment of schistosomiasis are the same as those already described, and include OZ03, OZ05, OZ10, OZ11, OZ15, OZ19, OZ23, OZ24, OZ25, OZ27, OZ31, OZ32, OZ40, OZ41, OZ43, OZ44, OZ47, OZ48, OZ61, OZ63, OZ70, OZ71, OZ77, OZ80, OZ83, OZ84, and OZ89. Most preferred compounds are OZ10, OZ11, OZ15, OZ23, OZ25, OZ27, OZ31, OZ41, OZ43, OZ47, OZ63, OZ71, OZ77, Z78, and OZ89. Preferred dosing levels of the spiro and dispiro trioxolanes are about 100-200 mg/kg/day orally.
The Spiro and dispiro trioxolanes of this invention may also have effectiveness in the treatment of cancer. Compounds having an endoperoxide moiety that is reactive with heme and iron have shown an ability to kill cancer cells. (See e.g. U.S. Pat. No. 5,578,637, the disclosure of which is hereby incorporated by reference). As noted with respect to artemisinin, trioxolanes"" mechanism of action against malarial parasites is based on the ability of trioxolane compounds to react with the iron in free heme molecules in malaria parasites, with the generation of free radicals leading to cellular destruction. Similarly, trioxolanes are selective against cancer cells due to the higher concentration of transferrin receptors on cancer cell membranes that pick up iron at a higher rate than normal cells. In the presence of the trioxolanes of this invention, the cancer cells will accumulate high concentrations of free radicals, leading to cell death. For cancer treatment, the trioxolanes of this invention may be administered in the doses and manner outlined above.
Other drugs besides trioxolanes which are compatible with the carrier ingredients may also be incorporated into the carrier. Such drugs may be readily ascertained by those of ordinary skill in the art and may include, for instance, antibiotics, other antimalarials, antiinflammatory agents, etc.
It is understood that the present invention contemplates the use of not only the above-stated trioxolane compounds themselves, but their prodrugs which metabolize to the compound and the analogues and biologically active salt forms thereof, as well as optical isomers which provide the same pharmaceutical results.
The following examples are offered to illustrate but not limit the invention. Thus, they are presented with the understanding that various formulation modifications as well as method of delivery modifications may be made and still be within the spirit of the invention.